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This assay consists of measuring the difference in factor V activity (prothrombin time assay) before and after incubation of a mixture of normal plasma and patient's plasma for 1 hour at 37 degrees C. For optimal sensitivity, the factor V value of the normal plasma is adjusted to approximately 20%, because the factor V assay is more sensitive in this area of the curve. In addition, an excess of patient's plasma will make the test more sensitive to small amounts of inhibitors.(Owen CA Jr, Bowie EJW, Thompson JH Jr: The Diagnosis of Bleeding Disorders. 2nd edition. Boston, MA, Little, Brown and Company, 1975, pp 143-145)

If the inhibitor screen is positive for an inhibitor of factor V, the inhibitor will be quantitated by the "Bethesda assays." In the Bethesda procedure, inhibitors are quantified by mixing equal volumes of serially diluted plasma with normal plasma. This mixture is incubated 2 hours at 37 degrees C, and its factor V activity is measured and compared to a control run at the same time. The difference between the factor V activity of the patient's incubation mixture and that of the control is used to calculate titer. The residual factor V activity is converted to "Bethesda units": 50% residual factor V is equal to 1 Bethesda unit.(Kasper CK, Aldedort LM, Counts RB, et al: A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh 1975;34:869-872)